1. Field of the Invention
The present invention provides kits for determination of N-acetyl-.beta.-D-glucosaminidase (hereinafter referred to as NAGase) activity, which is one of the important markers of renal dysfunction and methods for determining the activity by using them.
2. Prior Art
4-Methylumbelliferyl N-acetyl-.beta.-D-glucosaminide (hereinafter referred to as 4MU-NAG) and p-nitrophenyl N-acetyl-.beta.-D-glucosaminide (hereinafter referred to as PNP-NAG) have long been known as substrates for determining NAGase activity. The methods by using them have, however, such disadvantages that they require urine blanks and substrate blanks in each test, they are liable to be affected by inhibitors in urine, and they need a long time for the determination.
Sodio-meta-cresolsulfonphthaleinyl N-acetyl-.beta.-D-glucosaminide (hereinafter referred to as MCP-NAG), wherein meta-cresolsulfonphthaleine (hereinafter referred to as MCP) is a color indicator, was developed and disclosed in KOKAI 58-994 as a substrate capable of reducing such disadvantages as above. It still has, however, disadvantages in that it requires a substrate blank and a terminating step to determine NAGase activities with addition of an alkaline agent (one-point method).
Recently, 2-chloro-4-nitrophenyl-N-acetyl-.beta.-D-glucosaminide (hereinafter referred to as CNP-NAG) was developed and disclosed in KOKAI 61-112092 as a reagent for determining NAGase activity without termination of the reaction (continuous method). In this method, enzyme activity is monitored continuously and the method can be easily adapted to automatic analysers. This still has a disadvantage, however, in that it takes several minutes to dissolve CNP-NAG completely, even in the presence of surfactants, because the substrate is hardly soluble in water. Further, it takes much time to determine the activity of a trace amount of NAGase.